CN117756912A - Composite cytokine and application thereof in preparation of striae gravidarum repair emulsion - Google Patents
Composite cytokine and application thereof in preparation of striae gravidarum repair emulsion Download PDFInfo
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- CN117756912A CN117756912A CN202311779644.0A CN202311779644A CN117756912A CN 117756912 A CN117756912 A CN 117756912A CN 202311779644 A CN202311779644 A CN 202311779644A CN 117756912 A CN117756912 A CN 117756912A
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Abstract
The invention provides a composite cytokine and application thereof in preparation of striae gravidarum repair emulsion, and the provided repair emulsion can effectively repair striae gravidarum. The provided composite cytokine consists of natural killer cytokine solution and mesenchymal stem cell MSC cytokine solution. The composite cytokine prepared by the invention contains the combination of immunocytoactive factors and stem cell active factors, can participate in immunity and repair, eliminates damaged cell tissues, and has the functions of repairing skin injury and promoting regeneration. The striae gravidarum repairing emulsion containing the composite cytokines can mobilize skin immunity and repair, provide moist epidermis environment for skin repair, reduce skin texture formation, improve skin elasticity and reduce striae gravidarum. The invention has safe raw materials and obvious effect, and can be used in pregnancy and lactation period with confidence.
Description
Technical Field
The invention belongs to the technical field of beauty and health care products, and particularly relates to a composite cytokine and application thereof in preparation of striae gravidarum repair emulsion.
Background
Stretch marks are stretch marks developed during pregnancy and are mainly distributed on the abdomen, buttocks, chest, back and limbs, and are a common pathological change of skin in gestation period, and the skin is early represented by dark red or purple red stripes, and then pigment is deprived, atrophic and stabilized as white or silver stripes. Stretch marks are usually flat and normal in width at the beginning, but become wider over time. Striae gravidarum is mainly caused by physical stretching, pregnancy related hormones, structural changes of dermal collagen and elastic tissues, fibroblast activity, protein catabolism and other factors. The striae gravidarum can not be reduced after being formed without treatment, and although the striae gravidarum does not have threat to the health of patients, psychological diseases can be caused due to the influence on the beauty of the skin.
Since the pathological mechanism of striae gravidarum is not yet studied clearly, the treatment means such as external medicine, acupuncture and instruments with limited striae gravidarum are usually accompanied by corresponding side effects. It was found that the elastic fibers of the dermis superficial layer break and become rare in early stage of striae gravidarum lesions, and collagen fibers separate and become homogenously deformed, vessel wall thickens, lumen distensions, perivascular oedema, lymphocyte infiltration. Therefore, one possible therapeutic direction for striae gravidarum is to mobilize the self-healing ability of the body, help the body clear diseased cells in time, repair damaged tissues, increase skin elasticity, and maintain skin function and health.
Disclosure of Invention
The invention aims to provide a composite cytokine and application thereof in preparation of striae gravidarum repair emulsion, and the provided repair emulsion can effectively repair striae gravidarum.
The invention provides a compound cytokine, which is composed of natural killer cell (NK cell) factor solution and mesenchymal stem cell MSC cytokine solution;
the NK cell factor solution is obtained by separating human umbilical blood mononuclear cells (CB-MNC) from umbilical blood, and the umbilical blood mononuclear cells and trophoblast cells are co-cultured by using a serum-free culture medium added with IL-2 and a nutritional supplement; filtering the supernatant fluid by a filter membrane after the culture is completed, and concentrating filtrate to obtain NK cell factors;
the concentration of the nutritional supplement is 1-10% in a serum-free culture medium, and the nutritional supplement comprises human transferrin, human albumin, human platelet lysate, recombinant human insulin and D-galactose;
wherein the weight portions of the components are as follows: 5 to 10 parts of human transferrin, 50 to 100 parts of human albumin, 1 to 50 parts of human platelet lysate, 5 to 10 parts of recombinant human insulin and 1 to 5 parts of D-galactose.
The filter membrane is sequentially filtered by filter membranes with molecular weights of 50KD and 3 KD.
The culture is carried out at 37 ℃ with 5% CO 2 Culturing under the condition that the serum-free culture medium containing IL-2 is added every 2-3 days in the culture process, and the cell concentration is maintained to be not lower than 5 multiplied by 10 5 cells/ml, all cell suspensions were harvested on day 14 of culture.
The MSC cytokine solution is formed by mixing MSC cell supernatant and MSC cell lysate.
The MSC cytokine solution is prepared by culturing umbilical cord mesenchymal stem cells by using a serum-free culture medium, subculturing, and collecting the supernatant of a P3-P5 generation mesenchymal stem cell culture solution and umbilical cord mesenchymal stem cells; crushing and cracking the collected umbilical cord mesenchymal stem cells to obtain a cell lysate; mixing the cell lysate with the supernatant of the stem cell culture solution, and filtering the mixed solution with a filter membrane to prepare MSC cytokine solution;
the filter membrane filtration is to firstly use a 50KD filter membrane for filtration, then use a 3KD filter membrane for concentration, and finally use a 0.22 mu m filter membrane for sterilization and filtration to obtain MSC cytokine solution.
Further, the volume ratio of the NK cytokine solution to the MSC cytokine solution is 1:1;
furthermore, the compound cell factor is also added with any one or more of D-trehalose, chitosan oligosaccharide, sodium hyaluronate, lysine, glycine, arginine, vitamin E and vitamin B.
The complex cytokine, one specific description of the examples is as follows: the NK cell factor solution and the MSC cell factor are taken according to the volume ratio of 1:1 to be compounded, and 5 percent of D-trehalose, 2 percent of sodium hyaluronate, 0.05 percent of lysine, 0.05 percent of glycine, 0.05 percent of arginine, 1 percent of vitamin E and 1 percent of vitamin B are added.
The composite cytokine provided by the invention is used for preparing emulsion products for repairing striae gravidarum.
In a further aspect the invention provides an emulsion preparation for repairing stretch marks, the preparation comprising a complex cytokine as described above.
The composite cytokine prepared by the invention contains the combination of immunocytoactive factors and stem cell active factors, can participate in immunity and repair, eliminates damaged cell tissues, and has the functions of repairing skin injury and promoting regeneration. The striae gravidarum repairing emulsion containing the composite cytokines can mobilize skin immunity and repair, provide moist epidermis environment for skin repair, reduce skin texture formation, improve skin elasticity and reduce striae gravidarum. The invention has safe raw materials and obvious effect, and can be used in pregnancy and lactation period with confidence.
Drawings
Fig. 1: example 1 figure of the effect of single and complex cytokines on human skin fibroblast proliferation activity;
fig. 2: example 1 figure of the effect of single and complex cytokines on human skin fibroblast proliferation;
fig. 3: example 1 Effect of Single and Complex cytokines on collagen content of human skin fibroblast
Fig. 4: example 2 skin texture image change patterns before and after striae gravidarum repair lotion treatment.
Detailed Description
The invention constructs a compound preparation for treating striae gravidarum from the perspective of organism immune repair.
Natural killer cells (Natural killer cell, NK) are a component of the innate immunity of the body, and NK cells can synthesize and secrete a variety of cytokines including interferon (IFN- γ), tumor necrosis factor (TNF- α), granulocyte-macrophage colony-stimulating factor (GM-CSF), anti-inflammatory cytokines (IL-10, IL-5), chemokines (CCL 3, CCL4, CCL5, XCL 1), and the like.
Mesenchymal stem cells (Mesenchymal stem cells, MSC) are an adult stem cell derived from the early endoderm with multipotent differentiation potential. MSCs secrete cytokines, cell growth factors, anti-apoptotic factors, etc., such as Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), nerve Growth Factor (NGF), hepatocyte Growth Factor (HGF), etc., by paracrine action.
For better illustrating the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples, but the embodiment of the present invention is not limited thereto.
Example 1: preparation method of composite cytokine
1. Preparation of NK cytokine solution
Recombinant K562 cells inactivated with hypochlorous acid stock solution were used as trophoblasts, isolated human cord blood mononuclear cells (CB-MNC) were subjected to cell counting and cultured with GT-T551H3The trophoblast cells were added in a 1:1 ratio based on the weight of the suspension. Taking 30mL of fresh cord blood as an example, adding lymphocyte separation liquid for centrifugation to obtain human cord blood mononuclear cells (CB-MNC) with a concentration of about 5×10 7 co-culturing umbilical cord blood mononuclear cells and trophoblast cells in proportion, and adding 200U/mL IL-2 and 10% nutritional supplement into serum-free medium (Takara serum-free cell culture medium GT-T551H 3); the nutritional supplement comprises human transferrin, human serum albumin, human platelet lysate, recombinant human insulin, and D-galactose; the weight portions of the components are as follows: 5 to 10 parts of human transferrin, 50 to 100 parts of human albumin, 1 to 50 parts of human platelet lysate, 5 to 10 parts of recombinant human insulin and 1 to 5 parts of D-galactose. The cells were placed at 37℃in 5% CO 2 Culturing in an incubator. After that, fresh serum-free culture medium containing IL-2 and nutrient supplement are added every 2-3 days to maintain the cell concentration at 5X 10 5 Above cells/mL, about 2000mL of total cell suspension was harvested on day 14 of culture. After centrifugation (3000 rpm/min,6 min) of the cell suspension, all cells and culture supernatant were collected separately. The purity of NK cells (CD 3-CD16+56+) is more than 80% through flow detection. About 1800mL of the supernatant was filtered with filters having molecular weights of 50KD and 3KD in order, concentrated, sterilized with a 0.22 μm filter to give about 100mL of NK cell factor solution, and stored at 4 ℃.
Detecting IFN-gamma, TNF-alpha, GM-CSF and IL-4 levels in the filtrate by using an ELISA method, which is strictly performed according to the instructions of a kit (Nanjing Ornitofu Co.); the concentration levels of the components are shown in Table 1.
Table 1: content table of NK cytokine components
The results show that the prepared NK cell factor contains more IFN-gamma, TNF-alpha, GM-CSF, IL-4 and other components, can stimulate the proliferation of skin fibroblasts, promote the recombination and regeneration of broken elastic fibers and collagen fibers, and can up-regulate the gene expression of type I collagen and type III collagen, so that the collagen fibers are more organized.
2. Preparation of MSC cytokine solution
Serum-free medium was prepared and serum substitutes (glutamine, transferrin, vitamin C, insulin, progesterone, cortisol, human serum albumin) were added to the alpha-MEM medium. The culture medium is used for culturing the P3 generation mesenchymal stem cells by 1.5X10 7 For example, 30mL of serum-free medium was used to re-suspend the cells, and the cells were placed at 37℃in 5% CO 2 Culturing in an incubator. And (3) subculturing when the growth density of the cells reaches 80% -90%, digesting with 0.25% pancreatin, taking cell suspension, centrifuging (1000 rpm/min,5 min), separating supernatant and stem cells, continuously subculturing the stem cells, and reserving the supernatant at 4 ℃. Collecting all the mesenchymal stem cell culture supernatant of P3-P5 generation about 400mL and obtaining about 2X 10 cells 8 Cells.
After the stem cells are washed by normal saline, the stem cells are placed in liquid nitrogen/37 ℃ for freeze thawing, and the process is repeated for three times for 3min each time. After lysis, the cells were further lysed using an ultrasonic disruptor for 5s with 5s intervals, and 3 runs were performed. Centrifuging (1000 rpm/min,5 min) at 4deg.C, and collecting supernatant to obtain stem cell lysate. Mixing cell lysate with supernatant of stem cell culture solution, centrifuging at 4deg.C (3000 rpm/min,10 min), filtering with filter membrane with molecular weight of 50KD, collecting filtrate, centrifuging (10000 rpm/min,10 min), concentrating the secondary filtrate with 3KD filter membrane, sterilizing with 0.22 μm filter membrane to obtain MSC cytokine solution of about 50mL, and storing at 4deg.C.
The detection of VEGF, bFGF, IL-13, TGF-beta and KGF levels in the filtrate by ELISA was performed strictly according to the instructions of the kit (Nanjing Orthododofovir). The concentration levels of the components are shown in Table 2.
Table 2: MSC cell factor each component content table
The results show that the prepared MSC cytokines contain more VEGF, PDGF, bFGF, IL-13, TGF-beta, KGF, IGF, IL-1 and other components, promote proliferation and division of fibroblasts, epithelial cells and the like, regulate production of plasmin activators, promote synthesis and deposition of collagen, improve skin elasticity, reduce pigmentation and repair skin injury.
3. Preparation of composite cytokines
Taking 10mL of NK cell factor solution and MSC cell factor solution according to the volume ratio of 1:1, adding 5% of D-trehalose, 2% of sodium hyaluronate, 0.05% of lysine, 0.05% of glycine, 0.05% of arginine, 1% of vitamin E and 1% of vitamin B, and the balance of physiological saline, uniformly mixing, and carrying out low-temperature freeze-drying to obtain the composite cell factor.
And performing a skin fibroblast proliferation influence test by using the prepared composite cytokine. The lyophilized powder of the complex cytokine is diluted with PBS to a cytokine dilution concentration of 1 mg/mL. Selecting primary human fibroblast with good growth, digesting, and preparing into about 1X with cell culture solution
10 4 cell suspensions of cells/mL were inoculated into 96-well plates, 200. Mu.L of cell culture medium was added to each well, 200. Mu.L of cell culture medium was added to the peripheral wells, and the wells were placed in a cell culture tank and incubated at constant temperature and humidity for 24 hours. The randomization was divided into blank, solvent, MSC, NK and mixed factor groups. 6 duplicate wells per component. Adding 10 μl of sterile PBS solution containing 2% FBS into each well of solvent group cell, adding 10 μl of MSC cytokine solution into each well of MSC factor group cell, adding 10 μl of NK cytokine solution into each well of NK factor group cell, adding 10 μl of composite cytokine diluent into each well of mixed factor group cell, placing 96 well plate at 37deg.C, and 5% CO 2 Culturing in an incubator. The growth and proliferation of cells were detected by MTT method at 24h, 48h and 72h of culture, respectively. Add 20. Mu.L of MTT solution 5g/L per well, incubate in incubator for 4h, then remove the original medium, add 150. Mu.L of dimethyl sulfoxide (DMSO) per well, shake on shaker for 10min at low speed. Detecting Optical Density (OD) at 492nm wavelength of enzyme-linked immunosorbent assay, calculating cell proliferation activity,the calculation formula is as follows:
cell viability = test group mean OD/blank mean OD x 100%.
The viability and proliferation rate of fibroblasts determine the firmness of the skin and thus affect the condition of the skin. The viability of fibroblasts is reduced and the number is reduced, resulting in skin aging. Thus, the compaction efficacy of the sample can be evaluated by testing the effect of the sample on proliferation of human fibroblasts. The results show that the cytokines in the test group can promote cell proliferation, and the proliferation activity rate is higher than that of the compound cytokine group when the test group is cultured for 24 hours, 48 hours and 72 hours. When the cell proliferation activity of the compound cytokine group reaches 157% during 72 hours of culture, the NK factor group and the MSC factor group are 91% and 81% respectively, which shows that the compound cytokine can remarkably improve the cell proliferation activity (figure 1). The nucleus of the composite factor group is positioned at the center and is in oval and vortex migration, the cells are full, and the growth confluence of the fibroblast is high, which shows that the composite factor can promote the proliferation of the skin fibroblast more effectively than the single factor, and the proliferation rate of the cell is obviously improved (figure 2).
And performing a skin fibroblast collagen content influence test by using the prepared composite cytokine. The lyophilized powder of the complex cytokine is diluted with PBS to a cytokine dilution concentration of 1 mg/mL. The density of fibroblast HSF with good growth state is regulated to be 1 multiplied by 10 5 cells/ml, inoculated in 12-well plate, 1 ml/well, cultured for 24h, after cell monolayer is attached, the culture solution is discarded. The randomization was divided into control, MSC factor, NK factor and mixed factor groups. 4 duplicate wells per component. Adding cell culture solution containing 1% PBS into each cell of control group, adding cell culture solution containing 1% MSC cytokine solution into each cell of MSC factor group, adding cell culture solution containing 1% NK cytokine solution into each cell of NK factor group, adding cell culture solution containing 1% composite cytokine diluent into each cell of mixed factor group, placing 24 pore plate at 37deg.C, and 5% CO 2 Culturing in an incubator. Incubation was continued for 24h, cells were scraped off in PBS and the cell content was collected by centrifugation and ultrasonication for detection. By light splittingPhotometry, measuring the content of hydroxyproline at the wavelength of 560nm, and converting the content of collagen in cells according to the content of 14% of hydroxyproline in the collagen.
The content of collagen determines the thickness of collagen bundles, and influences the arrangement rule of collagen, thereby leading to striae gravidarum. Therefore, regulating collagen metabolism is an effective method for preventing striae gravidarum. The results showed that the cytokines of the test group each increased collagen production by fibroblasts, with hydroxyproline content of 2.03 μg/ml, MSC cytokine group of 1.20 μg/ml and NK cytokine group of 1.36 μg/ml, indicating that the composite cytokine significantly increased collagen production by fibroblasts compared to one cytokine alone (fig. 3).
Example 2: preparation method of striae gravidarum repair emulsion containing composite cytokines
1. Weighing the following components in parts by weight: 5% of compound cytokine powder, 5% of glycerol, 3% of shea butter, 2% of pentanediol, 1.5% of chitosan oligosaccharide, 1% of cetostearyl olive oleate, 0.5% of soybean lecithin, 0.02% of acetyl tetrapeptide-2, 0.01% of palmitoyl tripeptide-5 and the balance of purified water.
2. Mixing the shea butter, the cetostearyl olive oleate and the soybean lecithin, and stirring for 20min at 50-60 ℃ to obtain an oil phase; taking water, glycerol, pentanediol, chitosan oligosaccharide, acetyl tetrapeptide-2 and palmitoyl tripeptide-5, and stirring at 50-60 ℃ for 20min to obtain a water phase. Mixing the water phase and the oil phase, homogenizing and stirring for 10min, cooling to room temperature, adding the composite cytokine powder, and continuing homogenizing and stirring for 20min. The striae gravidarum repairing emulsion containing the composite cytokines is prepared.
3. The striae gravidarum repair emulsion containing the composite cytokines prepared by the invention is used for carrying out tests of moisture content of human skin cuticle, percutaneous moisture loss and skin elasticity, and scoring the striae gravidarum before and after treatment. And (5) performing skin imaging retention on the striae gravidarum of the abdomen before and after treatment, and comparing treatment effects.
The human subject: women aged 22-50 years are selected, and after 2 years of production, the women have striae gravidarum, are not allergic to common cosmetics, and do not participate in other clinical researches for nearly three months.
Detection instrument: the host of the Cotometer MPA580 (Courage+Khazaka, germany) was equipped with a skin moisture content Cornemeter CM825 test probe, a skin moisture loss Tewater-ter TM300 test probe, and a skin elasticity PVM600 test probe.
Detection conditions: the indoor environment is stable, the temperature is 20+/-2 ℃, and the environment humidity is 40-60%.
Detection time: 8 weeks.
The using method comprises the following steps: screening qualified subjects, performing positioning test on elasticity and texture of the abdomen, and photographing after stretch mark position measurement. The subject continues to use the test sample at home. The striae gravidarum part is continuously used for 8 weeks and 1 time daily without other striae gravidarum removing modes. After the test is completed, the test subject is tested.
Detecting the index: data statistics, expressed as standard error of Mean (mean±se, n=20), were compared by taking relative values, relative values (%) = (each measurement value ∈initial measurement value) ×100%. The data are analyzed and processed by SPSS20.0 software, the score of striae gravidarum before and after treatment is measured data, the score is expressed by mean ± standard deviation, the difference comparison adopts t test, and P < 0.05 is statistically significant.
Table 3: striae gravidarum condition scoring standard table
Detection result: the results showed that there was a significant change in skin stratum corneum moisture content, transdermal moisture loss and skin elasticity starting at week 2 and increasing stratum corneum moisture content by 17.5% by the end of week 8 experiments; the percutaneous water loss was gradually decreased, and the relative value of the percutaneous water loss was decreased by 8.2% at week 8 (Table 4).
Table 4: skin index change result table after treatment
Striae gravidarum condition scores were significantly reduced following treatment with striae gravidarum repair emulsions containing the complex cytokines, striae gravidarum color, severity, depth, and skin texture scores were significantly less than prior to treatment, with differences statistically significant (P < 0.05) (table 5).
Table 5: score comparison result table of striae gravidarum before and after treatment (X+ -S, score)
| Color of | Degree | Depth of | Texture of | |
| Before treatment | 3.15±0.47 | 3.02±0.22 | 3.58±0.45 | 2.88±0.59 |
| After treatment | 1.57±0.7* | 1.94±0.34* | 1.35±0.76* | 1.06±0.31* |
Note that: * P < 0.05 compared with the prior treatment
The skin imaging results showed that the image contrast found that the texture of the skin was lighter, the striae of pregnancy was lighter, and the color was similar to normal skin (fig. 4).
In conclusion, the composite cytokine prepared by the invention can participate in immunity and repair, remove damaged cell tissues, and has strong functions of repairing skin injury and promoting regeneration.
Claims (10)
1. A composite cytokine, wherein the composite cytokine comprises a natural killer cytokine and a mesenchymal stem cell cytokine;
the natural killer cell factor is obtained by separating human cord blood mononuclear cells from cord blood, and the cord blood mononuclear cells and trophoblast cells are co-cultured by using a serum-free culture medium added with IL-2 and a nutritional supplement; filtering the supernatant fluid with a filter membrane after the culture is completed, and concentrating filtrate to obtain natural killer cell factors;
the mesenchymal stem cell factor is formed by mixing MSC cell supernatant and MSC cell lysate.
2. The complex cytokine of claim 1, wherein the nutritional supplement comprises human transferrin, human albumin, human platelet lysate, recombinant human insulin, and D-galactose.
3. The composite cytokine of claim 1, wherein the filtration with the filter membrane is performed sequentially with a filter membrane having a molecular weight of 50KD and a filter membrane having a molecular weight of 3 KD.
4. The composite cytokine according to claim 1, wherein the mesenchymal stem cell factor is obtained by culturing umbilical cord mesenchymal stem cells in serum-free medium and subculturing, and collecting the supernatant of the P3-P5 generation mesenchymal stem cell culture solution and umbilical cord mesenchymal stem cells; crushing and cracking the collected umbilical cord mesenchymal stem cells to obtain a cell lysate; mixing the cell lysate with the supernatant of the stem cell culture solution, and filtering the mixed solution with a filter membrane.
5. The composite cytokine according to claim 4, wherein the filter membrane is a 50KD filter membrane, a 3KD filter membrane, and a 0.22 μm filter membrane.
6. The composite cytokine according to claim 1, wherein the volume ratio of NK cytokine to mesenchymal stem cell cytokine is 1:1.
7. The composite cytokine according to claim 1, wherein the composite cytokine is further added with any one or more of D-trehalose, chitosan oligosaccharide, sodium hyaluronate, lysine, glycine, arginine, vitamin E, and vitamin B.
8. The compound cytokine according to claim 1, wherein the compound cytokine is prepared by mixing NK cytokine solution and MSC cytokine in a volume ratio of 1:1, and adding 5% d-trehalose, 2% sodium hyaluronate, 0.05% lysine, 0.05% glycine, 0.05% arginine, 1% vitamin E, 1% vitamin B.
9. Use of the composite cytokine of claim 1 for the preparation of a preparation for repairing stretch marks.
10. An emulsion product for repairing stretch marks, wherein the product comprises the composite cytokine of claim 1.
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| CN202311779644.0A CN117756912A (en) | 2023-12-22 | 2023-12-22 | Composite cytokine and application thereof in preparation of striae gravidarum repair emulsion |
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| CN202311779644.0A CN117756912A (en) | 2023-12-22 | 2023-12-22 | Composite cytokine and application thereof in preparation of striae gravidarum repair emulsion |
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